1. Styryl molecules exhibit a range of cell-associated fluorescence patterns
We began by assessing how variation in chemical structure of styryl molecules influenced the intracellular intensity and distribution of the molecules' fluorescence signal in relation to Mitofluor™ Green —a lipophilic cation used as a mitochondria-specific fluorescent marker (50
). At the total intensity level, the distribution of fluorescence signal acquired from the styryl molecules was similar to that of Mitofluor™ Green controls (). However, the ratio of cytoplasmic-to-nuclear fluorescence of styryl molecules was considerably lower than that of Mitofluor™ Green (), consistent with fluorescence signals being more nuclear, more diffuse and coming from sites other than mitochondria. Also, the coefficient of variation (CV) of intracellular fluorescence of styryl molecules was lower than that of Mitofluor™ Green (), indicative of a more homogenous, intracellular fluorescence localization.
Figure 1 The distributions of whole cell intensity (A), cytoplasmic to nuclear ratio (B), and whole cell coefficient of variation (C), along with representative images (1-6) from various points along each distribution. All images are composites acquired from the (more ...)
Upon visual inspection, images of cells incubated with different styryl molecules revealed different patterns of intracellular fluorescence signal intensity and localization consistent with the measured image features (). Those images of cells with low cytoplasmic to nuclear ratio (, image 1) generally had a diffuse cellular staining pattern that appeared in both nuclear and cytoplasmic regions. Images of cells with intermediate cytoplasmic-to-nuclear ratio (, image 2) often had strong cytplasmic fluorescence suggestive of mitochondrial or some other cyplasmic organelle accumulation, and often exhibited some punctate nuclear (nucleolar) fluorescence. Images of cells with high cytoplasmic-to-nuclear ratios (, image 3) most closely resembled Mitofluor™ Green staining, in terms of the preferential localization of probe fluorescence in the cytoplasmic compartment and visual resemblance to the typical, perinuclear localization pattern of mitochondrial-specific fluorescent dyes.
A high value for the CV feature is an indicator of heterogenous staining patterns associated with vesicular or organellar dye sequestration (26
). Yet, a significant number of styryl molecules exhibited lower CV values than Mitofluor™ Green indicative of diffuse staining. Images at the extreme, low end of the CV values often had cells with diffuse, cytoplasmic fluorescence (, image 4). Images of cells with intermediate CVs exhibited various localization patterns, from membrane-associated (, image 5) to more punctate or vesicular staining patterns typical of mitochondrial or lysosomal staining. Images with the highest CVs of cell-associated fluorescence signals corresponded to cells with small dye crystals in the perinuclear region (, image 6).
We noted that the variance of cytoplasmic-to-nuclear ratios () obtained from the Mitofluor™ green controls was greater than the distribution observed for styryl compounds. This result was paradoxical at first, since the styryl molecules correspond to a diverse collection of compounds that should label cells differently, while Mitofluor™ Green is a single compound that should label all cells the same. However, we found that because the total nuclear signal of Mitofluor™ Green was very low, small variations in nuclear intensity could lead to large differences in the calculated cytoplasmic-to-nuclear ratio. In the case of styryl molecules, the nuclear and cytoplasmic signals are more similar to each other, so larger variations in nuclear signal of styryl molecules have a smaller effect on the cytoplasmic-to-nuclear ratio.
2. Phenotypic effects that confound analysis of subcellular localization features are associated with specific molecules
In any collection of prospective bioimaging agents, some phenotypic effects resulting from probe accumulation inside cells and non-intended interaction with cellular components can be expected. Because toxic effects generally become apparent after prolongued incubation with probes, we kept incubation times at the minimum. However, some styryl molecules did cause cell shape changes which may be indicative of probe toxicity (, image 4). Cell rounding and blebbing was specifically observed with particular aldehydepyridinium building block combinations. For example, cell incubated with probes D132 and E132 were rounded, with E132 showing signs of blebbing. Yet, many cells incubated with the closely-related probes A132, B132, D22 and E22 showed no signs of cell rounding or blebbing (). For quantitative structure-localization relationships, images of cell populations exhibiting signs of toxicity were excluded from analysis.
Figure 2 Images of cells incubated with a group of related styryl probes, exhibiting normal (A132, B132, D22 and E22) and rounded (D132, E132) cell shape phenotypes. All images are composites acquired from the Hoechst™ (blue) and TRITC channel (yellow, (more ...)
A different kind of phenotypic effect was associated with the appearance of cell-associated dye precipitates or crystals (, image 6). Crystals could be identified based on their extremely punctate and bright signal, as well as the rod-, star- or rhombus-shape of the particles (). Some of these crystals could be observed in cell-free, extracellular regions of the images. Yet, in many cases these crystals were closely associated with the individual cell nuclei and led to very high CV values as artifact (, image 6). We found that these insoluble styryl molecules were mostly associated with specific aldehyde groups, independently from the pyridinium or quinolinium group. These aldehydes were 87, 88, 124 and 127. As with dye-induced cell rounding, images with evidence of dye crystals in the immediate nuclear periphery (as in ) were excluded from further analysis.
Figure 3 Images of cells exhibiting cell associated insoluble dye aggregates or crystals, in the presence of styryl molecules possessing the same aldehyde (87) but different pyridinium or quinolinium building blocks (A-H). All images are composites acquired from (more ...)
To test the stability of the probe fluorescence pattern, the cytoplasmic-to-nuclear ratio and CV was compared in the presence and in the absence of extracellular probe (following prior incubation of cells with probes). Comparing these two conditions, the correlation for cytoplasmic-to-nuclear ratio was 0.64 and the correlation for CV was 0.92. Therefore, the staining patterns observed for most probes appeared quite stable, in steady state vs. efflux conditions. These trends were confirmed by visual inspection of the corresponding images (data not shown).
3. Different isomers of the pyridinium or quinolinium building block yield different subcellular fluorescence localization patterns
Because the chemical fingerprint used to calculate the Tanimoto coefficient is only sensitive to the presence or absence of a particular functional group in a molecule, many isomers of molecules possess a Tanimoto coefficient of 1.0 and therefore represent the most structurally similar pairs of molecules in a library. If subcellular localization of a molecule is governed by non-specific physicochemical properties of the probes (like probe radius, lipophilicity, number of hydrogen bonds, etc.), then subcellular probe signal should show minimal variation among isomers, compared to modifications that alter the chemical structure of the molecules by adding or subtracting atoms or functional groups.
In the styryl library, 6 out of the 8 pyridinium or quinolinium building blocks, and 55 out of 168 aldehyde building blocks possess structural isomers (). Unexpectedly, different isomers of the quinolinium building blocks often possessed different subcellular localization patterns (). These patterns were consistent and associated with a specific isomer. For example, the fluorescence signal of molecules D63, D72, D71 and D69 is present over the nuclear and cytoplasmic regions of the cell to similar extent, and they all exhibit a similar, heterogenous membrane-staining pattern. In contrast, the corresponding isomers E63, E72, E71, and E69 exhibit a distinctly bright and diffuse cytoplasmic staining with much darker nuclei. We note that varying the length of the hydrocarbon chain associated with aldheyde building block 63, 72, 71, to 69 is expected to increase the lipophilicity of the molecules by more than two orders of magnitude (data not shown). This suggests that the quinolinium D and E building block isomers exerts a far more prominent effect on subcellular localization than a 100-fold change in lipophilicity. Frequency histogram plots of CV and nuclear-to-cytoplasmic ratio features support this observation: group D produced far more compounds with high CV compared to group E, whereas groups D and E were very similar in terms of their cytoplasmic-to-nuclear ratios (data not shown).
Figure 4 Isomers of aldheyde building blocks (indicated by numbers separated by commas) and pyridinium or quinolinium building blocks (indicated by letters separated by commas) in the styryl library. Note that some aldheydes have two or three related isomers. (more ...)
Figure 5 Image pairs of cells incubated with styryl molecules synthesized with the same aldehyde building block (63, 72, 71 and 68) but different quinolinium building blocks (D and E). All images are from the TRITC channel (1s exposure with extracellular dye). (more ...)
4. Different isomers of the aldehyde building block yield different subcellular fluorescence localization patterns
Like the isomers of the pyridinium or quinolinium building block, isomers of the aldehyde building block () that shared the same pyridinium or quinolinium building block often exhibited different patterns of cell-associated fluorescence (). For example, in molecule D141 the methyl group on the aldehyde building block is in the ortho position while in the in molecule D143 it is in the para position. Yet, the signal of molecule D141 is associated with the heterogeneous, staining across the whole cell, while that of the molecule D143 is located in nucleoli as well as being diffusely localized in the rest of the cell ().
Figure 6 Image pairs of cells incubated with styryl molecules synthesized with the same quinolinium building blocks (D or E) but different aldheyde building blocks (141, 143, 31, 19, 131, 42). All images are from the TRITC channel (1s exposure with extracellular (more ...)
In another example of aldehyde building block isomers (), molecules E19 and E41 posses a methoxy group in ortho and para positions respectively, while molecules E42 and E131 possess a hydroxy group at the corresponding positions. In molecule E19 and E131 the fluorescence exhibits mitochondrial/cytoplasmic localization. However, in molecule E42 the fluorescence shows a punctate, cytoplasmic localization in some cells while in E41 the localization is in both nuclear or cytoplasmic region (). Thus very small variations in the structure of the aldehyde building block can lead to significant changes in the subcellular distribution of probe fluorescence.
5. Similar pairs of styryl probes generally yield similar spectral and intensity signals but dissimilar fluorescence localization patterns
Paired cheminformatic-machine vision analysis was performed to study how variation in fluorescence signals acquired from pairs of styryl probes was related to variation in their chemical structure. Three different analysis were performed: (1) total intensity analysis, which involved comparing the total signal in FITC, TRITC or Cy5 channels; (2) spectral analysis, which involved comparing the fraction of the total fluorescence signal that is obtained from each channel; (3) spatial analysis, which involved comparing the cytoplasmic-to-nuclear ratio and coefficient of variation of fluorescence signal in each channel.
As expected, a clear relationship between chemical similarity between each pair of probes in the library and their relative fluorescence in FITC, TRITC and Cy5 channels was observed. For pairs of styryl probes sharing the same aldehyde building block but different pyridinium or quinolinium building blocks, the more similar the molecules (higher Tanimoto coefficient) the more similar the fraction of total signal acquired in each of the three fluorescence channels (). A similar trend was observed in terms of the relative signals obtained in each of the three channels (). However, for pairs of probes sharing the same pyridinium or quinolinium groups but different aldehydes, the trend was not as prominent, either for the total signal intensity () or for the spectral analysis (). This indicates that variation in the pyridinium or quinolinium building blocks exerts a stronger effect on the fluorescence of the styryl molecules in FITC, TRITC and Cy5 channel than variation of the aldehyde, with the more similar building blocks leading to more similar intensity and spectral signals. The calculated correlation coefficients between image features similarity and chemical feature similarity support these trends ().
Figure 7 The pairwise dissimilarity in the total signal intensity (A, C) or the spectral distribution of signal intensity (B, D) plotted against standardized Tanimoto similarity between each pair of chemical structures in the styryl library. Pairwise comparisons (more ...)
In contrast, image feature analysis of cell-associated fluorescence signals revealed no visually obvious trend relationship between similarities in the chemical structure of each pair of probes, and the cytoplasmic-to-nuclear ratio () or CV () of cell-associated probe fluorescence. Thus, for every pair of molecules in the library, pairs of molecules that are similar to each other (based on their Tanimoto coefficient) did not necessarily exhibit similar localization of fluorescence signal compared to less similar pairs of probes, independent of whether the pairwise analysis was done across different pyridinium or quinolinium () or different aldehyde () building blocks. This result indicates that the mechanism leading to differences in nuclear-to-cytoplasmic probe distribution or CV values is less dependent on structural features captured by the chemical fingerprint of the molecules, compared to the mechanism leading to differences in the spectral distributions or total fluorescence intensity in the FITC, TRITC and Cy5 channels. This is consistent with our other observations ( and ) that small changes in the structure of the molecules - such as ortho vs. para isomers- exert a major effect on their subcellular localization features. Again, these observations correspond to the correlation coefficients between image features similarity and chemical feature similarity ().
Figure 8 The pairwise dissimilarity in the log cytoplasm-to-nucleus ratio (A, C) or the CV of cell-associated fluorescence intensity (B, D) plotted against standardized Tanimoto similarity between each pair of chemical structures in the styryl library. Pairwise (more ...)